What are the four stages of the cell cycle? Which stages comprise interphase?
- G1: growth phase preceding DNA synthesis - S: DNA synthesis - G2: growth phase preceding division - M: mitosis = cell division • G0 = differentiated cells that have exited the cell cycle • Interphase = G1, S and G2
Discuss checkpoints of cell
- G1/S = restriction point – checking if conditions are favorable for division - G2/M = has DNA been replicated and are conditions still favorable for division - Metaphase/anaphase transition point = are c/s all attached to mitotic spindles
The cell cycle is driven by the activity of cyclin-dependent kinases. Which cyclin-CDK complexes are most important at: a.) M-phase b.) Mid G1 phase c.) Late G1 phase d.) S phase
1.) M-phase: cyclin A/CDK1 and cyclin B/CDK1 2.) Mid G1 phase: cyclin D/CDK4 and cyclin D/CDK6 3.) Late G1 phase: cyclin E/CDK2 4.) S phase: cyclin A/CDK2
What is meant by the term contact inhibition?
- Mammalian cells don’t divide unless signaled to do so - Cell/cell interactions generally block inhibition = contact inhibition
What are mitogens? How do they function. Provide examples of these
- These are tissue-specific growth factors that are required to stimulate division, they allow cells to pass the restriction point, stimulate early response genes such as c-Fos, which stimulate transcription of delayed response genes, such as cyclin D, CDK4/6, cyclin E and CDK2 - Examples: PDGF, EGF, NGF, IGF-1, IGF-2
How do growth factors stimulate cell division? Discuss growth factor binding and all intracellular pathway
1.) Binding growth factor stimulates dimerization and autophosphorylation on the tyrosine residues of the receptors (growth factor receptors contain intrinsic protein tyrosine kinase activity). 2.) Phosphorylation residues form docking sites*. 3.) Adaptor protein GRB2 binds phosphotyrosine, which recruits guanine nucleotide exchange factor Sos, which activate 4.) Sos activates g-protein Ras by promoting exchange of GDP (Ras + GDP = inactive) for GTP (Ras + GTP = active) 5.) Ras then activates Raf (aka MAP kinase kinase kinase) 6.) Raf activates MAP kinase kinase (aka MEK) by phosphorylating it 7.) MAP kinase kinase activates MAP kinase through phosphosphorylations, which translocates into the nucleus to regulate transcription of many genes, including stimulation transcription of gene encoding c-Fos 8.) c-Fos stimulates transcription of cyclin D, CDK4/6 9.) Cyclin D, CDK4/6 phosphorylates Rb (retinoblastoma) in Rb:E2F complex, causing dissociation of Rb from E2F. 10.) E2F is a transcriptional activator of cyclin E/CDK2 (late G1 cyclin), which also acts to phosphorylate Rb:E2F as in 9. E2F also is a transcriptional activator of cyclin A (s-phase cyclin) * Most signal Ras/MAP kinase pathway
Why is phosphorylation of Rb such as crucial event in cell cycle progression? Which kinases phosphorylate Rb?
- Phosphorylation of Rb in Rb:E2F by cyclin D:CDK4/6 causes dissociation of Rb from E2F - E2F is a transcriptional activator of cyclin E:CDK2 - In the absence of growth factors, Rb phosphorylation can still occur independent of cyclin D:CDK4/6 and cell cycle can progress
How is CDK4/6 activity inhibited
- INK4 proteins inhibit these proteins. - INK4 transcription is stimulated by growth inhibitory factors
Describe regulation of G1/S transition
- S phase requires active cyclin A-CDK2, which is initially formed as inactive complex with inhibitor called p27kip1 - Late g1: cyclin E-CDK2 is desphosphorylated by phosphatase (Cdc25A) - cyclin E-CDK2 phosphorylates p27kip1 in complex w/ cyclin A-CDK2, cyclin A-CDK2 is activated by being freed from p27KIP2 - It promotes DNA replication by phosphorylating components of ori complexes
How is entry into mitosis coupled to completion of DNA replication?
- Completion of DNA replication activates protein phosphatase (cdc25C) - Protein phosphatase dephosphorylates a particular tyrosine residue in CDK1, which activates cyclin A or B-CDK1 complexes - Cyclin A or B-CDK1 complexes phosphorylate many proteins that participate in early mitotic events including chromosome condensation, assembly of mitotic spindle and breakdown of nuclear membrane
Would a normal cell divide if chromosomes were not correctly attached to the mitotic spindle? Explain
- No - Anaphase of mitosis is delayed until chromosomes attach to mitotic spindle - Correct assembly of mitotic spindle activates anaphase promoting complex (APC) - APC allows degradation of cohesin complexes, which hold sister chromatids together at centromere, sister chromatids can move to opposite poles - APC acquires different subunit and causes degradation of cyclin A or B
Discuss how proteins can return to their interphase states after the M phase
- APC causes degradation of cyclin A and B, which shuts off CDK1 activity and stops mitotic events from occurring - Desphosphorylation of cyclin-CDK targets by Cdc14 returns proteins to their interphase state: promotes decondensation of chromatin, disassembly of mitotic spindle, reassembly of nuclear membrane, cytokinesis) - APC becomes inactivated and Rb is dephosphorylated
Describe ways by which ATM/ATR kinases can prevent cell cycle progression in the face of DNA damage.
1.) ATM is activated by double-stranded breaks 2.) ATR is activated by UV light and certain DNA-damaging drugs - Both kinases stabilize p53 - P53 upregulates p21cip1 - p21 inhibits activates a kinase that prevents cdc25a and cdc25C from activating cyclin A/E-CDK2 (S phase) and cyclin A/B-CDK1 (M phase) respectively. - ATM and ATR also activate a checkpoint kinase that prevents cdc25a and cdc25C from activating cdc25a and cdc25C from activating cyclin A/E-CDK2 (S phase) and cyclin A/B-CDK1 (M phase) respectively
How is ATM activated? Implication
- ATM is activated by replication forks: when RFs are present, DNA replication is present and cell-cycle needs to be halted until this completes - ATM also activated by double-stranded breaks
Describe role of ATM in G2/M checkpoint
1.) ATM kinase senses replication forks, which indicates ongoing replication, and prevents checkpoint kinase from allowing activation of Cdc25C, which normally dephosphorylates CDK1. Therefore CDK1 remains inactive. ATM remains active as long as replication forks active
What is ataxia telangiectasia?
- Rare inherited disorder resulting from mutations in gene that encodes ATM kinase - Symptoms: difficulty in coordinating movement (ataxia) and appearance in small, widened blood vessels on skin (telangiectasia) - Pts susceptible to infections and at increased risk of leukemias and lymphomas. They are highly sensitive to radiation exposure.
What is meant by the term apoptosis? What are the two pathways?
- Apoptosis = programmed cell death, orderly series of events leading to cell death and destruction 1.) Intrinsic pathway = activation of p53 2.) Extrinsic pathway = death receptors
What enzymatic activity do the caspases possess?
- destroy cellular components, degrade nuclear lamina, trigger nuclear/cell fragmentation
In general terms, describe how activation of p53 leads to efflux of cytochrome c from the mitochondria, ultimately resulting in caspase activation and cell death.
- ATM/ATR activity stabilizes p53 (remember, ATM and ATR are activated by double-stranded DNA breaks and UV light/damaging drugs respectively) - Targets of p53 include proapoptotic proteins belonging to Bcl-2 family - Bcl-2 family proteins (not Bcl-2, which is antiapoptotic) stimulates BAX activity, which promotes release of cytochrome c from mitochondria - Cytochrome c binds to Apaf-1 in cytoplasm - Oligomerization of cyt c with apaf-1 occur - This recruits caspase 9, initiator caspase - Caspase 9 activates executioner caspases, such as caspase 3 - Cell is destined to death